Detection of Thyrotropin-Receptor Messenger Ribonucleic Acid ...

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The Journal of Clinical Endocrinology & Metabolism 89(8):3705–3709 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-031967

Detection of Thyrotropin-Receptor Messenger Ribonucleic Acid (mRNA) and Thyroglobulin mRNA Transcripts in Peripheral Blood of Patients with Thyroid Disease: Sensitive and Specific Markers for Thyroid Cancer PRIYA CHINNAPPA, LESLIE TAGUBA, ROSEMARIE ARCIAGA, CHARLES FAIMAN, ALLAN SIPERSTEIN, ADI E. MEHTA, SETHU K. REDDY, CHRISTIAN NASR, AND MANJULA K. GUPTA Departments of Endocrinology, Diabetes and Metabolism (P.C., L.T., C.F., A.E.M., S.K.R., C.N.), Clinical Pathology (R.A., M.K.G.), and General Surgery (A.S.), The Cleveland Clinic Foundation, Cleveland, Ohio 44195 Because thyroid cancer cells express functional TSH receptors (TSHR), TSHR-mRNA in peripheral blood might serve as a tissue-/cancer-specific marker. We measured circulating TSHR-mRNA by RT-PCR in 51 normal controls, 27 patients with benign thyroid disease, 67 patients with treated differentiated thyroid cancer (DTC), and eight patients with newly diagnosed DTC, preoperatively. Results were compared with thyroglobulin (Tg) mRNA and serum Tg levels. TSHR-mRNA signals were not detected in normal controls and in 24 of 27 (89%) patients with benign thyroid disease. All 19 patients with treated DTC with evidence of distant or local disease tested positive for TSHR-mRNA (sensitivity 100%). Among patients with no evidence of disease, TSHR-mRNA was detected in 1 in 48 (specificity 98%). Six of the eight newly diagnosed DTC patients tested preoperatively were positive for TSHR-

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ONITORING FOR THYROID cancer recurrence after total thyroidectomy and radioactive iodine (RAI) ablation is routinely done through measurement of serum thyroglobulin (Tg) and 131I whole-body scanning (WBS) (1– 3). Measurement of serum Tg has proven to be a useful marker for residual disease, but its major limitations include a low sensitivity during thyroid hormone suppression therapy, as well as interference with the test by Tg autoantibodies (4, 5). Although the sensitivity of serum Tg to detect metastatic disease improves markedly after thyroid hormone withdrawal, this method induces symptomatic hypothyroidism, causing significant morbidity in many patients. In an attempt to circumvent these limitations, a number of investigators have used RT-PCR to detect cancer cells in the circulation by amplifying and detecting mRNA transcripts for thyroid-specific genes such as Tg, thyroid peroxidase, or sodium/iodide symporter (6 –9). Among these, Tg-mRNA Abbreviations: DTC, Differentiated thyroid cancer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RAI, radioactive iodine; rhTSH, recombinant human TSH; Tg, thyroglobulin; TSHR, TSH receptor; WBS, whole-body scanning. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

mRNA. The concordance between TSHR-mRNA and Tg-mRNA and between TSHR-mRNA and serum Tg was 95%. Fourteen patients with DTC (21%) had Tg antibodies, three with local disease (all positive for TSHR-mRNA), and 11 with no evidence of disease (all negative for TSHR-mRNA). Our results indicate that TSHR-mRNA and/or Tg-mRNA in peripheral blood are both equally sensitive and specific markers for monitoring thyroid cancer patients. Their principal value resides in the Tg antibody-positive patients in whom a positive or a negative mRNA value might have indicated or obviated the need for a whole-body scan. Furthermore, the high specificity combined with their ability to predict thyroid cancer preoperatively suggests a potential role in detecting thyroid cancer in patients with thyroid nodules. (J Clin Endocrinol Metab 89: 3705–3709, 2004)

has been the most studied marker (10 –14). However, there has been a great deal of variability and inconsistency among the results obtained in various studies. Some investigators failed to detect Tg-mRNA in patients with metastatic disease. Others (10, 11) detected it in the blood from healthy subjects (6, 7, 13, 14). Although attempts to quantify Tg-mRNA have been reported to find differences between healthy individuals and cancer patients, there remained a significant overlap between these groups. The variability in Tg-mRNA results in various studies may be explained by the alternative splicing of Tg-mRNA in thyroid cells, as at least 16 alternative splicing sites have been reported (15). Recently, Savagner et al. (16) measured the relative proportion of two alternative variants of Tg-mRNA in thyroid cancer and normal subjects. They showed that, with use of an appropriate cutoff level, Tg-mRNA was valuable in detecting recurrent thyroid cancer in patients taking l-T4 therapy. Nonetheless, in most studies the use of Tg-mRNA remained limited to the detection of recurrent disease in T4-treated thyroidectomized cancer patients (16 –19). Tg production by both normal and neoplastic thyroid tissues depends on the presence of functional TSH receptors (TSHR) and is influenced by TSH levels. The expression of TSHR in thyroid neoplasms has been well documented (20 –

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Chinnappa et al. • TSH-Receptor mRNA and Thyroid Disease

22). However, this marker has not been exploited for detection of thyroid cancer cells in blood. We have shown previously that detection of TSHR- as well as Tg-mRNA signals in normal healthy subjects was dependent on primer selection (23). Using these selected primers, we investigated the sensitivity and specificity of TSHR-mRNA and Tg-mRNA detection by RT-PCR in the peripheral blood from normal subjects and from patients with thyroid cancer and benign thyroid diseases. Subjects and Methods Subjects A total of 153 patients including 51 normal subjects without a history of thyroid disease (female to male ratio ⫽ 1.7; age range, 25– 60 yr), 27 with benign thyroid disease (female to male ratio ⫽ 3.5; age range, 18 –77 yr), and 75 patients with differentiated thyroid cancer (DTC) (female to male ratio ⫽ 3.2; age range, 20 – 80 yr) were evaluated. Among the 27 patients with benign thyroid disease, three patients had thyroiditis, 18 had solitary thyroid nodules or multinodular goiters, three had primary hypothyroidism on replacement T4 therapy, and three had Graves’ disease. Among the DTC patients, 67 (89%) had a near-total thyroidectomy, and 65 (83%) had had RAI ablation at least 1 yr before mRNA testing. The remaining eight patients, all with newly diagnosed papillary thyroid cancer, were tested before thyroidectomy. All DTC patients were evaluated during visits as outpatients in the Department of Endocrinology, Diabetes and Metabolism at the Cleveland Clinic Foundation. A chart review was conducted to obtain each patient’s history, operative/ pathology reports, and laboratory and radiological examinations. The pathology, disease status, treatment status, and Tg antibody status of 75 patients with thyroid cancer are listed in Table 1. Forty-nine patients were studied while on T4 suppression, seven after T4 withdrawal, and 11 after the administration of recombinant human TSH (rhTSH) (Thyrogen; Genzyme, Cambridge, MA). Among 49 patients evaluated during T4 therapy, 41 (86%) had a diagnostic RAI scan within 12 months from the date of testing, and four patients had a diagnostic RAI scan within 24 – 48 months before the testing; all were monitored with serum Tg determinations and were considered free of disease if the scan was negative and/or they had undetectable Tg levels. Four had no available scans, two of them had recurrent disease (one with pulmonary and one with node metastasis), one had negative ultrasonography and lymph node biopsies, and the last had undetectable serial serum Tg levels and no clinical evidence of disease. None of the patients received RAI therapy after their last WBS except in three with known metastatic disease as confirmed by other imaging procedures and pathological examination. Blood samples for TSHR- and Tg-mRNA were collected at various intervals from the initial date of surgery. Concurrent serum levels of TSH (Roche Diagnostics, Indianapolis, IN) and Tg (Nichols Institute Diagnostics, San Juan Capistrano, CA) were measured by immunochemiluminometric assay, and the sensitivity was defined as 1.0 ␮g/liter. Tg antibodies were also measured in most patients by enzyme immunoTABLE 1. Characteristics of thyroid cancer patients

Treatment status

Treated T4 therapy T4 withdrawal After rhTSH Newly diagnosed Presurgery Total

N

Tg Ab positive

Number of patients tested (number with disease) by pathological diagnosis Papillary Ca

Follicular Ca

Hu¨ rthle cell Ca

49 7 11

12 2 0

40 (12) 6 (1) 11 (0)

6 (3) 1 (0) 0 (0)

3 (3) 0 (0) 0 (0)

8 75

0 14

8 (8) 65 (21)

0 7 (3)

3 (3)

Ab, Antibody; Ca, carcinoma.

assay (Tosoh Bioscience, Inc., South San Francisco, CA). A Tg value of at least 1.0 ␮g/liter in patients tested during T4 therapy and a value of at least 2.0 ␮g/liter in patients tested after rhTSH administration or after T4 withdrawal was considered to be a significant indicator for follow-up WBS (24). All patients on T4 suppression had TSH values less than 1.0 mU/liter (61% had TSH ⬍ 0.1 mU/liter) except five with TSH levels between 1.0 and 2.8 mU/liter. All patients studied after T4 withdrawal had TSH values greater than 30 mU/liter except one (TSH of 19 mU/liter). Our RAI scanning procedure included measurement of neck uptake 24 h after administration of a tracer dose (100 ␮Ci) of 131I and a diagnostic WBS obtained at 48 h after administering a 5-mCi dose of 131I. Patients tested after rhTSH (0.9 mg im for 2 d) had a 5-mCi dose of 131I administered 24 h after the second dose of rhTSH and a WBS obtained 48 h thereafter. Blood samples for both TSH/Tg-mRNA as well as Tg measurement were drawn at the same time before the scan. Scans were reviewed by the nuclear medicine physicians and were considered positive if they showed visible uptake in the thyroid bed and/or discrete focal uptake was present at sites that normally did not pick up 131I. Patients were classified as having evidence of disease if they had a positive WBS or disease diagnosed by pathology or found by other nonradioiodine imaging modalities. The study was reviewed and approved by the Institutional Review Board of the Cleveland Clinic Foundation and written informed consent was obtained before enrollment into the study.

RT-PCR Approximately 3–5 ml of whole blood (collected in EDTA-treated tubes) was mixed with equal volume of PBS, pH 7.4, layered with 8 ml Ficoll (Pharmacia, Peapack, NJ) and centrifuged at 400 ⫻ g for 20 min at 4 C. The mononuclear cell layer was collected, washed, and pelleted. RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. OD ratio of A260/280 was used to assess the quality and quantity of isolated RNA. One to 2 ␮g total RNA was reverse-transcribed to cDNA using Superscript Pre-amplification System (Invitrogen) following the instruction manual. PCR was performed using the selected primer pairs as described previously (23). The primer sequences were: TSHR: forward 5⬘-GCTTTTCAGGGACTATGCAATGAA-3⬘ and reverse 3⬘-AGAGTTTGGTCACAGTGACGGGAA-5⬘ (212 bp); Tg: forward 5⬘-AGGGAAACGGCCTTTCTGAA-3⬘ and reverse 3⬘-CTTTAGCAGCAGAAGAGGTG-5⬘ (407 bp); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a control gene ubiquitously expressed, was also analyzed to confirm the success of RNA extraction and RT and PCRs using primers as previously reported (25). PCR was carried out for 38 cycles [94 C for 1 min (first cycle for 2 min), 62 C for 1 min, 72 C for 1 min (10 min for the last cycle)]. RT-PCR products were resolved on 2% gel electrophoresis and visualized by ethidium bromide staining. The estimated sensitivity for these assays was tested by serial dilution of thyroid cancer tissue RNA with RNA obtained from normal peripheral blood mononuclear cells (see Ref. 23 for details) and was found to be approximately 10 cancer cells per milliliter of blood.

Statistical analysis Data were analyzed for diagnostic sensitivity and specificity of TSHRmRNA and Tg-mRNA, serum Tg, and WBS for detection of recurrent/ residual thyroid cancer. The comparisons of sensitivity and specificity between the markers were performed using Fisher’s exact test. P ⬍ 0.05 was considered significant.

Results TSHR-mRNA in normal controls and benign thyroid disease

Results are summarized in Table 2. All 51 of the normal controls were negative for both TSHR- and Tg-mRNA. Of the patients with benign thyroid disease, 24 of 27 (89%) were negative for both TSHR- and Tg-mRNA, including 15 patients with thyroid nodules. Of the three positive patients,


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two had massive obstructive goiters (100 and 300 g; 10 ⫻ 8 ⫻ 3 cm and 11 ⫻ 11 ⫻ 5 cm, respectively), and the third had a follicular adenoma. All three patients had their diagnosis confirmed by surgical pathology. TSHR-mRNA in patients with thyroid cancer

Figure 1 shows the representative RT-PCR products for TSHR (212 bp), Tg (407 bp), and GAPDH (397 bp) as obtained in nine thyroid cancer patients, one normal subject, and one positive thyroid cancer control. Table 3 summarizes the results TABLE 2. TSHR mRNA and Tg mRNA positivity in healthy euthyroid subjects, subjects with benign thyroid disease, and thyroid cancer patients without evidence of disease Subjects

N

Normal Benign thyroid disease TC with no evidence of disease Total positives/total (% specificity)

51 27 48

No. positive (%) TSHR-mRNA

Tg-mRNA

Serum Tga

0 3 (11)b 1 (2)

0 3 (11)b 4 (8.5)

NA NA 2 (4)

4/125 (97%)

7/125 (94%)

NA, Not applicable. a Using a cutoff of at least 1 ␮g/liter on T4 therapy; at least 2 ␮g/liter after T4 withdrawal or rhTSH. b Two patients with massive obstructive goiters and one with a follicular adenoma.

obtained in the 67 previously treated DTC patients and in the eight patients with DTC tested before surgical resection. Forty-nine patients with TC were tested while on T4 therapy. Eight of these patients had distant metastases, 10 had local recurrences or cervical lymph node disease, and the remaining 31 were disease-free. Table 3 lists the percentage of positives as obtained with TSHR-mRNA, Tg-mRNA, serum Tg levels, and WBS. Both TSHR- and Tg-mRNA were positive in all patients with distant metastases or local disease. Also, serum Tg was detected all but one patient with recurrent disease while on T4 suppression. Three of seven follicular and all three Hu¨ rthle cell cancer patients with evidence of disease were positive for both TSHR- and Tg-mRNA. Of the 31 patients with no evidence of disease, TSHR-mRNA was detected in none and Tg-mRNA was detected in two (6%). These two patients had undetectable serum Tg values and negative WBS and remained disease-free at 1-yr follow-up. Seven patients were tested after T4 withdrawal. One had evidence of local recurrence on WBS and was positive for both TSHR- and Tg-mRNA. Of the six patients with no evidence of disease on WBS, one was positive for both TSHR- and TgmRNA but had an undetectable serum Tg value after T4 withdrawal. Another had a postwithdrawal serum Tg value of 55.5 ng/ml (negative antibodies; negative for TSHR- and TgmRNA) but 1 yr later was found to have an undetectable Tg value, negative WBS, and negative thyroid ultrasound on T4 therapy. The cause of this isolated high Tg level is not clear. Eleven patients were tested after rhTSH. None had evidence of disease. One was positive for Tg-mRNA but negative for TSHR-mRNA and WBS. This patient had a serum Tg value of 2.3 ng/ml after rhTSH. One year later, the serum Tg after rhTSH was undetectable despite no treatment having been given. Subsequent WBS and three consecutive Tg levels after thyroid withdrawal have been negative so far, and the patient has been considered as having no evidence of disease. Tg-mRNA in patients with anti-Tg antibodies

FIG. 1. Representative gel picture showing RT-PCR results for Tg, TSHR, and GAPDH in nine patient samples (lanes 1–9), one negative control (no RT; lane 10), and one positive control (thyroid cancer tissue RNA; lane 11). Lanes 1, 5, and 6, Benign thyroid disease patients; lanes 2 and 4, thyroid cancer patients with no evidence of disease; lanes 3 and 7–9, thyroid cancer patients with evidence of disease.

Fourteen (21%) of the patients with DTC had anti-Tg antibodies. Eleven of the 14 patients had no evidence of disease; all had an undetectable serum Tg level (measured values are spuriously low in the presence of Tg antibodies), and all were negative for TSHR-mRNA. All three patients with local dis-

TABLE 3. TSHR-mRNA, Tg-mRNA, and serum Tg levels in patients with thyroid cancer Patients

Treatment On T4 suppression Distant metastasis Local metastasis No disease After T4 withdrawal Local metastasis No disease After rhTSH No disease No treatment presurgery

N

No. positive (%) TSHR-mRNA

Tg-mRNA

Tga

8 10 31

8 (100) 10 (100) 0 (0)

8 (100) 10 (100) 2 (6)

8 (100) 9 (90) 0

6 (75) 6 (75)b 0 (0)c

1 6

1 (100) 1 (17)

1 (100) 1 (17)

1 (100) 1 (17)

1 (100) 0 (0)

11 8

0 (0) 6 (75)

1 (9) 6 (75)

1 (9) NA

0 (0) NA

NA, Not applicable. a Using a cutoff of at least 1 ␮g/liter on T4 therapy; at least 2 ␮g/liter after T4 withdrawal or rhTSH. b n ⫽ 8. c n ⫽ 29.

131

I Scan

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ease were positive for both TSHR- and Tg-mRNA, including one that was negative for serum Tg. Diagnostic performance of TSHR-mRNA

Table 4 summarizes the diagnostic performance characteristics of TSHR-mRNA and compares these with Tg-mRNA, serum Tg levels, and WBS to detect recurrent/metastatic disease. There were no statistically significant differences among these markers, and both TSHR-mRNA and Tg-mRNA had equal sensitivity for detection of residual/recurrent disease (P ⫽ 0.209, Fisher’s exact test). Two patients with no evidence of disease (negative scan and subsequent undetectable Tg levels) had elevated serum Tg levels, one after T4 withdrawal and another after rhTSH, but were negative for both TSHR-mRNA and Tg-mRNA. A WBS was not done in four patients and was negative in three patients with evidence of disease [two with lung metastases and one with node metastases (sensitivity ⫽ 82%)]. The concordances between TSHR-mRNA and TgmRNA and between TSHR-mRNA and serum Tg (in Tg antibody-negative patients) were both 95%. Discussion

The major finding in the present report is that the presence of TSHR-mRNA signals in blood is specific for patients with thyroid cancer, being undetectable in healthy subjects and in the vast majority of patients with benign thyroid diseases. Furthermore, we demonstrated the high specificity of Tg-mRNA for thyroid cancer when carefully selected primers are used in the assay. Thyroid carcinomas are known to contain functional TSHR (20 –22). To date, this target has not been exploited for detection of circulating cancer cells, perhaps due to previous reports showing the presence of TSHR-mRNA transcripts in peripheral blood mononuclear cells (26) and other extrathyroidal tissues (27). The finding of these transcripts in extrathyroidal tissues can be explained by TSHR splice variants (15, 16, 28). TABLE 4. Diagnostic performances of TSH-R mRNA, Tg mRNA, serum Tg level, and 131I uptake/WBS in previously treated thyroid cancer patients A: TSHR mRNA positivity: comparison with Tg mRNA, serum Tg, and WBS No. positive/total (%) Patients

TSHR-mRNA

Evidence of 19/19 (100) disease No evidence of 1/48 (2) disease Concordance with TSHR mRNA

Tg-mRNA

Serum Tg

131 I uptake/ WBS

19/19 (100)

18/19 (95)

13/17 (76)

4/48 (8)

2/48 (4)

0/46 (0)

64/67 (95)

64/67 (95)

59/63 (94)

B: Performance characteristics Diagnostic performance (%)

Sensitivity Specificity PPVa NPVb Efficiency a b

TSHR-mRNA

Tg-mRNA

Serum Tg

100 98 95 100 98

100 92 83 100 94

95 96 90 96 94

PPV, Positive predictive value. NPV, Negative predictive value.

131

I uptake/WBS

83 100 100 94 95

Thus, selection of primers specific to thyroid cells is of paramount importance in the assay (23). There was a high concordance between the TSHR- and TgmRNA in our series. These Tg-mRNA findings contrast with a number of previous studies using both qualitative and quantitative RT-PCR, which detected Tg-mRNA signals in most normal subjects (6, 10, 11). As discussed previously, these differences are most likely due to the primer pair selection rather than differences in assay sensitivities or other variables in methodologies. Indeed, we previously detected signals in controls as well, using some primer pairs described in the literature (23). It is possible that, with certain primer pairs amplification of pseudogenes can give rise to false-positive results (28). A more likely explanation for this discordance is the limitation of PCR-based techniques in their capability of detecting alternative splice variants amplified by the selected primers (16, 23). Although other investigators (8, 10) have reported a much higher incidence (20 –33%) of Tg-mRNA positivity in patients with benign thyroid disease, we obtained false-positive results in only three of 27 (11%) patients. One of these false-positive cases was a patient who was found to have a follicular adenoma on surgical pathology. The differentiation between follicular adenoma and follicular carcinoma is, at present, only possible after surgical resection and formal histological examination. To date, there are no known markers that can distinguish follicular adenomas from cancer with certainty because, like follicular cancer, a significant number of follicular adenomas harbor Ras mutations (29) and show galectin-3 immunostaining (30, 31). Therefore, the finding of circulating thyroid cells in a patient with follicular adenoma is not unparalleled and favors the notion that some follicular adenomas may represent a premalignant stage of follicular carcinomas (32). The other two false positive results were in patients with massive goiters, indicating that such patients may have circulating thyroid cells. Alternatively, an occult DTC may have been overlooked in the pathology examination. Regardless, because most other benign nodules are negative and six of eight DTC patients tested preoperatively were positive, this test may have a potential use in the differential diagnosis of cancer from benign thyroid nodules preoperatively and certainly warrants further study. Our results indicate that TSHR-mRNA by RT-PCR is a highly sensitive and specific marker in monitoring patients for recurrent or metastatic thyroid cancer. Our data also show the high sensitivity and reliability of serum Tg levels to detect most recurrent disease while a patient is on T4suppressive therapy. Therefore, RT-PCR assay may not prove to be a cost-effective alternative for serum Tg levels as a first line of testing. Its value lies in patients in whom Tg measurements are not reliable due to the presence of interfering Tg antibodies, heterophile antibodies, or other factors. In our series, TSHR-mRNA or Tg-mRNA was detected in all patients with local or distant metastases who were tested while on T4 therapy or after thyroid hormone withdrawal. Serum Tg levels were elevated (ⱖ2.0 ng/ml) in all but one (Tg antibody-positive) of these patients in whom the need for WBS would have been indicated by positive mRNA testing. Furthermore, the finding of a negative TSHR- or Tg-mRNA signal might have obviated the need for a WBS in two Tgpositive patients and in all Tg antibody-positive patients with no evidence of disease.


Among the 48 DTC patients who had no evidence of disease, TSHR-mRNA was positive (2%) less often than TgmRNA (8%). None of these positive patients had the disease at a 1-yr review as evidenced by serum Tg levels and/or WBS. Nonetheless, these patients deserve to be monitored carefully because they may harbor microscopic disease that can be detected earlier by the Tg-mRNA assay. Unlike previous reports (13), which find Tg-mRNA only in patients with papillary carcinoma but not in other histological types, we found no differences in either Tg- or TSHRmRNA results based on tumor histology. Among our patients, three of seven follicular carcinoma patients and all three Hu¨ rthle cell carcinoma patients with evidence of disease were positive for both TSHR- and Tg-mRNA. In summary, the presence of either TSHR-mRNA or TgmRNA in peripheral blood is specific for the presence of residual/recurrent DTC disease and is as sensitive as serum Tg in monitoring Tg antibody-negative patients. However, in Tg antibody-positive patients with unreliable serum Tg values, TSHR-mRNA or Tg-mRNA surveillance may prove to be more cost-effective by obviating the need for a WBS in mRNA-negative patients. Furthermore, the high specificity of mRNA testing combined with our preliminary findings of its ability to detect thyroid cancer preoperatively suggest a potential role in screening patients with thyroid nodules.

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14. 15. 16. 17.

18. 19.

20.

Acknowledgments 21.

Received November 12, 2003. Accepted March 18, 2004. Address all correspondence and requests for reprints to: Manjula K. Gupta, Ph.D., Department of Clinical Pathology, L-30, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. E-mail: [email protected].

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